The African penguin (Spheniscus demersus) is a critically endangered species endemic to southern Africa. Limited information is available on the parasite diversity associated with the species in natural settings. This study explores the diversity and incidence of parasites associated with African penguins and their nests, and records the effect of host and environmental factors on parasite infestation. Ecto-, haemo- and helminth parasites were recorded from 210 adult birds, 583 chicks and 628 nests across five colonies (two mainland and three islands) along the south-western coast of South Africa, in 2016 and 2017. Mean nest density (total and active nests) and climate variables (temperature and precipitation) were obtained for each colony. Parapsyllus humboldti was the most abundant and prevalent ectoparasite on penguins and in nests (69.10 and 57.80%, respectively), while Piroplasmorida/Haemospororida (33.51%) and Cardiocephaloides spp. (56.17%) were the most prevalent haemo- and helminth parasites of penguins, respectively. In general parasite abundance and prevalence was significantly affected by penguin age (chicks vs adults), location (mainland vs islands), nest density (total and active nests) and season (spring vs autumn/winter). It is concluded that parasite infestations are structured and that penguin chicks at mainland colonies are more susceptible to parasite infestations during spring.