Purpose. To determine the efficacy of povidone-iodine (P-I) prophylaxis for ophthalmia neonatorum (ON) in Angola and to document maternal prevalence and mother-to-child transmission rates. Methods. Endocervical samples from mothers (n = 317) and newborn conjunctival smears (n = 245) were analysed by multiplex polymerase chain reaction (PCR) for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG). Newborns were randomized into a noninterventional group and an interventional group that received a drop of P-I 2.5% bilaterally after conjunctival smear collection. Mothers were trained to identify signs of ON and attend a follow-up visit. Results. Forty-two newborns had ocular pathology, and 11 (4.4%) had clinical signs of ON at the time of delivery. Maternal PCR was positive for MG (n = 19), CT (n = 8), and NG (n = 2). Six newborns were positive for CT (n = 4), MG (n = 2), and NG (n = 1). Mother-to-child transmission rates were 50% for CT and NG and 10.5% for MG. Only 16 newborns returned for follow-up. Conclusions. Lack of maternal compliance prevented successful testing of prophylactic P-I efficacy in ON prevention. Nevertheless, we documented the prevalence and mother-to-child transmission rates for CT, NG, and MG. These results emphasize the need to develop an effective Angolan educational and prophylactic ON program.