The effect of pefloxacin on the pharmacokinetics parameters of rifampicin in humans was investigated using plasma and saliva concentrations. Five healthy volunteers (4 male and 1 female), ages 20–35 years, each received 600 mg rifampicin alone, and after a 1-week drug washout period, 600 mg rifampicin plus 500 mg pefloxacin was administered with 350 mL of water. Plasma and saliva concentrations of rifampicin were measured at 7 different time intervals and different pharmacokinetics parameters calculated. Pefloxacin coadministered with rifampicin reduced plasma and saliva elimination half-life, peak plasma concentration, area under the concentration time curve, volume of distribution, minimum absorption time, absorption rate constant, and absorption half-life showed a significant increase (P < 0.05). Time to reach peak plasma concentration was not affected with or without pefloxacin. Pefloxacin increases bioavailability of rifampicin and hence extends its detection in the body as demonstrated by rifampicin being estimated at 24 hours when coadministered with pefloxacin, whereas at 24 hours, it was completely absent when administered alone.