Antibiotic resistance is a global threat to public health, leads to health and economic burden. Studies show that knowledge and attitude towards antibiotic use is poor, especially among rural residents. However, there is no study conducted on knowledge and attitude towards antibiotic use among the public in rural area Malaysia. The aim of this study is to assess knowledge and attitude on antibiotic use among public in rural area Batu Pahat, Johor. This was a cross-sectional study in which closed-ended questionnaires were distributed to 350 rural residents in Batu Pahat by convenience sampling method. The questionnaire was constructed into socio-demographic, antibiotic use and indication, knowledge and attitude towards antibiotic use. The most inappropriate knowledge responses were found for assumption about the effectiveness of antibiotics towards viral infection (69.1%), colds and coughs (57.7%). Only few were aware about the decrease in antibiotic effectiveness following antibiotic overuse (29.1%). Misuse antibiotics for cold (64.0%), expect antibiotics to be prescribed for common cold symptoms (52.6%) and discontinue antibiotics when start feeling better (63.4%) were the several highest inappropriate responses in attitude domain. Significant association was shown between both knowledge level and attitude level with age, gender, educational level, monthly income, occupation related to healthcare, family member’s occupation related to healthcare and most common location seek for healthcare. In conclusion, the rural residents demonstrated moderate knowledge (50%) but negative attitude (56.9%) towards antibiotic use. To cope with antibiotic resistance issue, proper planning on effective methods to promote appropriate use of antibiotics are necessary.