Background: India accounts for (4%) of all malaria cases worldwide. The World malaria report 2017 showed that, by 2016, global progress against malaria had stalled and was off track to meet the Global Technical Strategy milestones for 2020. Aims and Objectives: The aim of the study is to assess knowledge of rural medical officers (RMO) and health workers on malaria epidemiology, diagnosis, and treatment as per National Vector Borne Disease Control Program of India guidelines. Materials and methods: A descriptive, observational, and cross-sectional study was done among government health personnel’s using a semi-structured questionnaire. Results: In the present study, 258 health personnel’s participated comprising multipurpose health workers female (MPHW-F), multipurpose health worker male (MPHW-M), and RMO. The standardized score on knowledge of vector biology and epidemiology was 72% and 54% for RMO and (MPHW-M), respectively. On malaria diagnosis, MPHW-M has got median score (53%) as compared to RMO (46%). On national malaria drug policy, there is a marginal difference between median score of RMO (42%) and MPHW-M (40%). Overall, there is incomplete and poor knowledge of treatment of malaria among all health personnel’s with lack of concept about presumptive treatment. Training showed some positive impact on the knowledge of MPHW-F but no impact on MPHW-M. Graduates and experienced workers have got better knowledge than undergraduates and MPHW-M have more knowledge than MPHW-F. There is poor knowledge of reporting formats, and usage of rapid diagnostic kits among all the health personnels. There was no significant impact of education qualification, in service training and work experience on the knowledge of Male health workers. Conclusion: Training needs to be suitably tailored as there is a lot of scope of improvement in the knowledge regarding malaria diagnosis and treatment among health workers.