Background: Irrational use of antibiotics and wrong practicing behavior leads to the problem of antimicrobial resistance. This can be confronted by spreading awareness with the assistance of doctors in a community. Aims and Objectives: The purpose of our study is to evaluate the knowledge, attitude, and practicing behavior regarding personal use of antibiotics and awareness of antimicrobial resistance in urban and semi-urban population. Materials and Methods: This is a questionnaire-based cross-sectional study, conducted in July–September 2022 at Shadan Institute of Medical Sciences, Hyderabad. Participants were divided into (1) urban coming from Greater Hyderabad municipal corporation limits and (2) semi-urban coming from Rangareddy district. Institutional ethical committee approval was obtained before the study. After obtaining consent from 130 participants, regional and English language questionnaires were distributed, data were collected and analyzed. Results: The study showed a majority (61.5%) in 18–40 years age group with female predominance (63.8%). Most of them had formal education (73%) with a middle-class background (39.2%). The actual knowledge of participants seems to be accurate (96.9%) about antibiotics. Most of the participants reported inappropriate antibiotic use such as disposal (63.1%), skipped doses (61.5%), without medical advice (62.3%), increased dose duration (60.8%), and decreased dose duration (64.6%) drug intake as per their understanding. Cold and flu (45.4%) were an indication for an antibiotic prescription. According to majority, hospital hygiene (51.5%) must be focused to slow down the occurrence of antimicrobial resistance. A significant difference between urban and semi-urban community members in knowledge (χ2 = 47.250, P = 0.01), attitude (χ2 = 46.357, P = 0.01), and practicing behavior (χ2 = 21.373, P = 0.02) was noticed. Conclusions: Urban showed accurate knowledge toward antimicrobial resistance. Attitude and practicing behavior of semi-urban population was noteworthy on antimicrobial resistance. Awareness regarding the rational use of antibiotics needs to be imparted by doctors. They should address about it sensibly and timely in community to avoid misuse or overuse of antibiotics.