The biological effects of omega-3 and omega-6 fatty acids are determined by their mutual interactions. This interaction extremely affects various functions. Lower consumption of omega-3 during gestation leads to various disorders, even in hearing. We aimed to assess the effect of low omega-3/omega-6 ratios on auditory nerve conduction. In this experimental study, the auditory brainstem response test was performed on 24-day-old rat (n=14). The rats were divided into case (low omega-3/omega-6 ratio during gestation and lactation) and control groups. Variables such as P1, P3, and P4 absolute latency period, interpeaks (P3-P4, P1-P3, and P1-P4), and P4/P1 amplitude ratio were measured. We found an increased P4 omega-3/omega-6 ratio in the group with a low omega-3/omega-6 ratio (P<0.01). No significant difference was observed in the P1 and P3 absolute latency period between the studied groups (P>0.05). Also, no significant difference was observed between the groups with respect to the P1-P3 interpeak latency (IPL) periods (P>0.05); while the P1-P4 and P3-P4 IPLs were significantly increased in the group with a low omega-3/omega-6 ratio (P<0.05). The P4/P1 amplitude ratio significantly decreased in the group with a low omega-3/omega-6 ratio (P<0.05). Results confirmed the negative effects of low omega-3/omega-6 ratio on the auditory system and hearing.