Environment hygiene is important for preventing infection and promoting a healthier environment in which to live or work. The goal of this study was to examine the antimicrobial effects of Citrus aurantifolia (key lime) juice and aqueous extracts of Cinnamomum iners (cinnamon) bark and Citrus hystrix (kaffir lime) leaves on the kinetic growth of Pseudomonas aeruginosa and methicillin resistance Staphylococcus aureus (MRSA). Antimicrobial activity was quantitatively evaluated using spectrophotometry and viable cell counts versus bacterial growth time. The fomite surface samples that were used in the second experiment were chosen randomly from the laboratories. They were assessed both before and after intervention using a mixture of commercial disinfectant detergent and lime juice. In the kinetic growth study, the lime juice effectively eliminated P. aeruginosa and MRSA. The cinnamon bark extract was more effective at inhibiting P. aeruginosa than MRSA. The kaffir lime leaf extract demonstrated bacteriostatic activity for the first 60 min, which then weakened after 90 min for both bacteria. The lime juice extract and commercial disinfectant mixture effectively disinfected the fomites. Further studies of the use of key lime juice as a disinfectant in the hospital environment should be conducted, as C. aurantifolia exhibits antibacterial activities against endemic microbes.