Background: Antimicrobial resistance is a serious concern and life threat in public health globally. Gram-positive clinical isolates are one of the leading etiological agents showing drug resistance traits. This study aimed to detect the antimicrobial resistance pattern of clinically isolated Gram-positive bacterial isolates collected from various renowned diagnostic centers of Dhaka city, Bangladesh. Methods: A laboratory-based cross-sectional study was conducted at the Microbiological Laboratory of the Department of Microbiology, Stamford University Bangladesh. A total of 104 clinical specimens (wound swab, pus, blood, urine, ear discharge, nasal swab, sputum, nipple discharge, breast milk, catheter tip, umbilical swab, and throat swab) from different age groups were collected from some renowned diagnostic centers of Dhaka city for analyzing the cultural and drug sensitivity patterns from January 2023 to April 2023. According to the study plan, only the Gram-positive bacteria were collected and transported to the laboratory of the Department of Microbiology, Stamford University Bangladesh following WHO guidelines, and the antimicrobial susceptibility tests were carried out by Kirby–Bauer disk diffusion method and the results were interpreted as per CLSI guidelines, 2022. Results: This study highlights the presence of Staphylococcus aureus (65.4%), Streptococcus spp. (23.1%), and Enterococcus spp. (11.5%) as causative agents of Gram-positive bacterial infections from overall 104 clinical specimens. Most of our isolates were found multidrug-resistant. For S. aureus isolates, cefixime (100%); for Streptococcus spp., doxycycline (95.83%); and for Enterococcus spp., amoxicillin (100%), cefixime (100%), colistin (100%), and cotrimoxazole (100%) were found to be the most ineffective drug when compared to other antibiotics. Conclusion: Our study portrayed the remarkable antibiotic resistance feature of Gram-positive bacterial isolates which is a matter of public health concern. It is necessary to carry out a continuous assessment of antibiotic sensitivity patterns of resistant Gram-positive bacteria; otherwise, it will become an uncontrollable problem not only in Bangladesh but also worldwide in the near future.