Abstract Background Since the Coronavirus Disease 2019 (COVID-19) pandemic, awareness of infection prevention and control (IPC) has increased in primary care settings. This study aimed to examine behavioural determinants shaping IPC behaviour pre-, during, and post-pandemic among healthcare workers (HCWs) in general practices, to inform optimised IPC in primary care. Methods For this qualitative study, semi-structured in-depth interviews were conducted during two study periods: (1) pre-COVID-19 pandemic: July 2019-February 2020, with 14 general practitioners (GPs) and medical assistants, and (2) during the COVID-19 pandemic: July 2022-February 2023, with 22 GPs and medical assistants. The design was informed by behaviour change theories. Data were analysed using thematic analysis. Results Main themes were: (1) risk perception and IPC awareness, (2) attitudes towards IPC and professional responsibility, (3) decision-making process and risk considerations for IPC adherence, (4) social norm and social influence in GP practice team, and (5) environmental context and resource availability in GP practice. During the pandemic, risk perception and awareness of the importance of IPC increased compared to the pre-pandemic period. A consistent belief emerged that IPC is part of professional responsibility, while needing to be balanced with other aspects of patient care. Decision-making is dependent on the individual GP and mainly influenced by risk assessments and sustainability considerations. The social context in the practice team can reinforce IPC behaviours. GP practice building and layout, and limited IPC resource and material availability were reported as main barriers. Conclusions The theory-informed insights of this study can be used for targeted interventions to optimise IPC behaviour in general practices. Adopting multifaceted strategies to target the various determinants is recommended to sustain IPC, by implementing continuous education using tailored communication, integrating IPC in work routines and ...