Nigella sativa (NS) is a plant that has long been utilized in traditional medicine as a treatment for certain diseases. The aim of this work was to valorize the essential oil (EO) of this species by phytochemical analysis and antimicrobial and antioxidant evaluation. EO was extracted by hydrodistillation from the seeds of Nigella sativa (EO-NS). Phytochemical content of EO-NS was evaluated by use of gas chromatography coupled to mass spectrometry (GC-MS/MS). Antioxidant ability was in vitro determined by use of three assays: 2.2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing power (FRAP), and total antioxidant capacity (TAC) relative to two synthetic antioxidants: BHT and quercetin. Antimicrobial effect was evaluated against four clinically important bacterial strains (Staphylococcus aureus, ATCC 6633; Escherichia coli, K12; Bacillus subtilis, DSM 6333; and Proteus mirabilis, ATCC 29906) and against four fungal strains (Candida albicans, ATCC 10231; Aspergillus niger, MTCC 282; Aspergillus flavus, MTCC 9606; and Fusarium oxysporum, MTCC 9913). Fifteen constituents that accounted for the majority of the mass of the EO-NS were identified and quantified by use of GC-MSMS. The main component was O-cymene (37.82%), followed by carvacrol (17.68%), α-pinene (10.09%), trans-sabinene hydrate (9.90%), and 4-terpineol (7.15%). EO-NS exhibited significant antioxidant activity with IC50, EC50, and total antioxidant capacity (TAC) of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M1"> <mn>0.017</mn> <mo>±</mo> <mn>0.0002</mn> </math> , <math xmlns="http://www.w3.org/1998/Math/MathML" id="M2"> <mn>0.1196</mn> <mo>±</mo> <mn>0.012</mn> </math> , and <math xmlns="http://www.w3.org/1998/Math/MathML" id="M3"> <mn>114.059</mn> <mo>±</mo> <mn>0.97</mn> </math> mg EAA/g, respectively. Additionally, EO-NS exhibited promising antibacterial activity on all strains under investigation, especially on E. coli K12 resulting in inhibition diameter of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M4"> <mn>38.67</mn> <mo>±</mo> <mn>0.58</mn> </math> mm and a minimum inhibitory concentration (MIC) of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M5"> <mn>1.34</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL. Also, EO-NS had significant antifungal efficacy, with a percentage of inhibition of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M6"> <mn>67.45</mn> <mo>±</mo> <mn>2.31</mn> </math> % and MIC of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M7"> <mn>2.69</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL against F. oxysporum, MTCC 9913 and with a diameter of inhibition <math xmlns="http://www.w3.org/1998/Math/MathML" id="M8"> <mn>42</mn> <mo>±</mo> <mn>0.00</mn> </math> mm and MIC of <math xmlns="http://www.w3.org/1998/Math/MathML" id="M9"> <mn>0.67</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL against C. albicans. To minimize development of antibiotic-resistant bacteria, EO-NS can be utilized as a natural, alternative to synthetic antibiotics and antioxidants to treat free radicals implicated in microbial infection-related inflammatory reactions.