Carob-mediated calcium hydroxide nanoparticles (C-CaOH2 NPs) are a type of NPs, newly developed via a green synthesis method, that have demonstrated good in vitro biocompatibility. However, their antibacterial potential has not yet been explored. Both calcium hydroxide and carob are known for their antibacterial potency as bulk materials; thus, we hypothesized that C-CaOH2 NPs may exhibit promising antibacterial efficacy. This study aimed to examine the antibacterial activity of C-CaOH2 NPs against aerobic and anaerobic strains using zone of inhibition (ZOI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests. The results showed that the Gram-negative aerobic strains are more susceptible than the Gram-positive stains. The most susceptible bacteria were P. aeruginosa, E. coli, and S. aureus, in that order. Conversely, P. micra and E. faecalis were the least susceptible strains. The best recorded ZOIs were at 100 and 150 mg/mL concentrations in all bacteria, with the greatest diameter (11.7 ± 0.6 mm) exhibited in P. aeruginosa. Moreover, the MICs for aerobes were 3.12 mg/mL, except for E. faecalis (0.78 mg/mL) and K. pneumoniae (1.56 mg/mL). MBCs were 12.5 mg/mL for all except P. aeruginosa (3.12 mg/mL) and K. pneumoniae (6.25 mg/mL). The anaerobic strain P. micra exhibited the highest values for both MIC (15.62 mg/mL) and MBC (31.25 mg/mL). The current investigation revealed that C-CaOH2 NPs have intermediate and dose-dependent antibacterial activity that may have variable biomedical applications.