This study evaluates the ototoxicity of a potential novel ototopical antimicrobial, photoactivated methyl-δ-aminolevulinic acid (M-ALA). Ten CBA/J mice received intratympanic injections of 10 mM M-ALA and 640 nm light source illumination for 7 days, with contralateral ears receiving saline. Auditory-evoked brainstem response (ABR) thresholds (8, 16, 24, and 32 kHz) were determined preinjection and at 1, 30, and 90 days postinjection. Mean ABR thresholds were similar after intratympanic administration of M-ALA and saline (F ratio, 0.001; P = .971). ABR thresholds temporarily increased in both groups (F ratio, 28.52; P = .00) at day 1 postinjection of intratympanic treatments but returned to baseline at day 30 and 90. This temporary elevation was associated with tympanic membrane perforations and granulation tissue at the injection sites, which resolved by day 30 posttreatment. The preliminary findings indicate that intratympanic application of M-ALA with light activation over a 7-day course in a murine model does not produce measurable ototoxicity and is well tolerated.