Bio-enhancers are molecules without drug activity that augment the bioavailability of drugs in combination therapy. The aim of this study was to assess the bio-enhancing activity of bioactive fractions obtained from plant extracts on commonly used antibiotics. The effect of ethyl acetate extract of Cleome rutidosperma leaves on the antibacterial activities of amoxycillin and cloxacillin against Staphylococcus aureus NCIM 2614 and Pseudomonas aeruginosa NCIM 2653, respectively, was studied. Moreover, the antimicrobial activities of extracts of different parts of C. rutidosperma were evaluated against S. aureus, P. aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Candida albicans, and Aspergillus niger. Hot ethyl acetate leaves extract (HEALE) and antibiotics were tested individually and in combination. HEALE showed maximum activity against test organisms; the minimum inhibitory concentration (MIC) of HEALE was 600 µg/mL. The test organisms were found to be multidrug-resistant (MDR) strains. However, HEALE or HEALE in combination with antibiotics did not lead to the development of resistance. Phytochemical and chromatographic analyses of HEALE revealed the presence of antimicrobial and bio-enhancing constituents. Such findings herald the possibility of a potentially active bio-enhancer of plant origin.