Most infections due to intracellular bacteria respond poorly to antibiotic treatment, The chemical conditions within the subcellular site of bacteria may change antibiotic activity. Coxiella burnetii multiplies within phagolysosomes. The antimicrobial activity of antibiotics combined with the lysosomotropic agents amantadine (1 µg/mL), chloroquine (1 µg/mL), and ammonium chloride (1 mg/mL), which alkalinized Coxiella burnetii-containing phagolysosomes from pH 4.8 to 5.3, 5.7, and 6.8, respectively, was evaluated. Percentages of residual viable bacteria (RVB) in cell cultures were significantly reduced after exposure to combinations of doxycycline (4 µg/mL) with amantadine (RVB = 18.2% ± 8.7%, P < .05), chloroquine (RVB = 0.64% ± 0.38%, P < .01), or ammonium chloride (RVB = 0.29% ± 0.17%, P < .01); the same was seen with pefloxacin (1 µg/mL) with chloroquine (RVB = 27.6% ± 10.8%, P < .05) or ammonium chloride (RVB = 3.72% ± 1.1%, P < .05). Such bactericidal activity correlated with increased phagolysosomal pH, as determined by Pearson's correlation coefficient, suggesting that phagolysosomal alkalinization is critical for the bactericidal effect of antibiotics.