Background Preconception care comprises interventions that aim to identify and modify biomedical, behavioural and social risks to a woman's health or pregnancy outcome through prevention and management. Despite its importance, it is less routinely practiced in Nigeria and information on the knowledge and practice of preconception care among health workers at the community level is scanty. Hence, this study investigated healthcare workers' knowledge, perception and practice of preconception care in Ibadan, Nigeria. Methods A descriptive, cross-sectional research design, using non-probability sampling, was used to select 80 health workers across two local government areas in Ibadan to participate in this study. Four research questions with two null hypotheses were formulated to guide the study and a semi-structured questionnaire was distributed to gather data on the participants' knowledge, perceptions and practice of preconception care. The data were analysed using regression analysis, with P<0.05 used to indicate significance. Results The majority of respondents had fair knowledge of preconception care (53.8%), and a generally positive perception (74.3%), but poor practice (42.2%) of preconception care. Only 15.0% practiced preconception care every day when caring for women and couples in their communities. Knowledge of preconception care was positively associated with health workers' age (P<0.000), occupation (P=0.002) and years of experience (P<0.000). Conclusions Healthcare workers in Ibadan need to be trained on preconception care guidelines and resources should be made available for their use at every level of healthcare delivery in Nigeria.