Purpose The aims of the study were to examine the acoustic reflex screening and threshold in healthy neonates and those at risk of hearing loss and to determine the effect of birth weight and gestational age on acoustic stapedial reflex (ASR). Method We assessed 18 healthy neonates (Group I) and 16 with at least 1 risk factor for hearing loss (Group II); all of them passed the transient evoked otoacoustic emission test that assessed neonatal hearing. The test battery included an acoustic reflex screening with activators of 0.5, 1, 2, and 4 kHz and broadband noise and an acoustic reflex threshold test with all of them, except for the broadband noise activator. Results In the evaluated neonates, the main risk factors were the gestational age at birth and a low birth weight; hence, these were further analyzed. The lower the gestational age at birth and birth weight, the less likely that an acoustic reflex would be elicited by pure-tone activators. This effect was significant at the frequencies of 0.5, 1, and 2 kHz for gestational age at birth and at the frequencies of 1 and 2 kHz for birth weight. When the broadband noise stimulus was used, a response was elicited in all neonates in both groups. When the pure-tone stimulus was used, the Group II showed the highest acoustic reflex thresholds and the highest percentage of cases with an absent ASR. The ASR threshold varied from 50 to 100 dB HL in both groups. Group II presented higher mean ASR thresholds than Group I, this difference being significant at frequencies of 1, 2, and 4 kHz. Conclusions Birth weight and gestational age at birth were related to the elicitation of the acoustic reflex. Neonates with these risk factors for hearing impairment were less likely to exhibit the acoustic reflex and had higher thresholds.