The emergence of beta lactamase producing bacterial strains eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Beta lactam antibiotics can be coupled with non-antibiotic adjuvants to combat these multidrug resistant strains. We study the synergistic antibiotic effect of stigmasterol as adjuvant of ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model. All test bacteria were beta lactamase producing clinical isolates. The combination showed significantly better antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity, and the effect was observed in both Gram positive and Gram negative bacteria. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the low fractional inhibitory concentration (FIC) index on Checkerboard Assay. The results suggest that the combination of ampicillin and stigmasterol acts additively in the treatment of infections caused by beta-lactamase producing pathogens. In bacterial growth reduction assay, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative to ethanol control. Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts at least by 98.7%. In conclusion, we found synergistic effects of stigmasterol and ampicillin against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant.