The surging trend of (re)emerging diseases urges for the early detection, prevention, and control of zoonotic infections through the One Health (OH) approach. The operationalization of the OH approach depends on the contextual setting, the presence of the actors across the domains of OH, and the extent of their involvement. In the absence of national operational guidelines for OH in India, this study aims to identify potential actors with an attempt to understand the current health system network strength (during an outbreak and non-outbreak situations) at the local health system of Ahmedabad, India. This case study adopted a sequential mixed methods design conducted in two phases. First, potential actors who have been involved directly or indirectly in zoonoses prevention and control were identified through in-depth interviews. A network study was conducted as part of the second phase through a structured network questionnaire. Interest and influence matrix, average degree, network density, and degree of centralization were calculated through Atlas.Ti (ATLAS.ti Scientific Software Development GmbH, Berlin, Germany), UCINET (Analytic Technologies, Lexington, KY, USA) software. The identified actors were categorized based on power, administrative level (either at the city or district level), and their level of action: administrative (policy planners, managers), providers (physicians, veterinarians), and community (health workers, community leaders). The matrix indicated that administrative actors from the district level were ‘context setters’ and the actors from the city level were either ‘players’ or ‘subjects’. The network density showed a strength of 0.328 during the last outbreak of H5N1, which decreased to 0.163 during the non-outbreak situation. Overall, there was low collaboration observed in this study, which ranged from communication (during non-outbreaks) to coordination (during outbreaks). The private and non-governmental actors were not integrated into collaborative activities. This study concludes that not only collaboration is needed for OH among the sectors pertaining to the human and the animal health system but also better structured (‘inter-level’) collaboration across the governance levels for effective implementation.