This study investigates the green synthesis of silver nanoparticles (AgNPs) using Ocimum gratissimum root extract, along with their characterization and antibacterial efficacy. Proximal analysis revealed low moisture (8.02 ± 0.11%), crude protein (3.31 ± 0.28%), crude fat (2.01 ± 0.59%), moderate fibre (9.18 ± 0.40%), and ash (5.11 ± 0.91%), indicating a carbohydrate-rich composition. AgNPs were synthesized using the root extract as reducing and stabilizing agent; UV–Vis spectroscopy confirmed their formation with a surface plasmon resonance peak at 425 nm, consistent with spherical nanoparticles (30–100 nm). FTIR identified phenolic and polysaccharide functional groups (O–H, C=O, C–O), while XRD indicated a face-centered cubic (FCC) structure with good crystallinity. SEM micrographs showed mostly spherical particles with minimal aggregation. Antimicrobial activity at 10–20 mg/mL was tested against Proteus mirabilis, Pseudomonas aeruginosa, Bacillus cereus, Aspergillus fumigatus, Candida albicans, and Saccharomyces cerevisiae. The root extract alone showed no activity at 10 mg/mL but exhibited dose-dependent inhibition at higher concentrations (ZOIs: 10.40–15.16 mm). AgNPs displayed stronger broad-spectrum efficacy (ZOIs: 7.12–18.9 mm), often surpassing gentamicin. These findings highlight the potential of O. gratissimum root-derived AgNPs as eco-friendly antibacterial agents against multidrug-resistant organisms.