Self-emulsified nanoemulsions (SENs), one of the promising lipid-based drug delivery systems may be used to deliver drugs through vaginal route. Vaginal cavity remains healthy because of the defensive action by its microflora against the pathogenic infections, and any disturbance to this microflora by the delivery systems gives invitation to the infections. In the present study, the growth inhibition and cytotoxic effects of two SENs and their components on L. acidophilus were evaluated. The two SENs showed inhibitory effects on the growth of L. acidophilus in a concentration-dependent manner when tested at the concentration range of 0.1-5.0%. The SEN composed of medium chain mono/di-glyceride had greater inhibitory effect than the one composed of long chain monoglyceride. The study on the effect by the individual lipids with the surfactant Kolliphor RH40 further confirmed that the growth inhibitory and cytotoxic effects were in the order of Capmul® MCM > Maisine® CC > Miglyol® 810 > Kolliphor® RH40. Both OD and CFU counting were used to measure the viability of the culture. The results from the two methods were in good correlation except when there was no growth, suggesting OD can be used when there is no complete growth inhibition.Copyright © 2020. Published by Elsevier Inc.