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Background: Informal and unregulated rural medical practitioners (RMPs) provide healthcare services to about two-thirds of people in Bangladesh, although their service is assumed to be substandard by qualified providers. As the RMPs are embedded in the local community and provide low-cost services, their practice pattern demands investigation to identify the shortfalls and design effective strategies to ameliorate the service. Methods: We conducted a cross-sectional study in 2015–16 using a convenient sample from all 64 districts of Bangladesh. Personnel practising modern medicine, without any recognized training, or with recognized training but practising outside their defined roles, and without any regulatory oversight were invited to take part in the study. Appropriateness of the diagnosis and the rationality of antibiotic and other drug use were measured as per the Integrated Management of Childhood Illness guideline. Results: We invited 1004 RMPs, of whom 877 consented. Among them, 656 (74.8%) RMPs owned a drugstore, 706 (78.2%) had formal education below higher secondary level, and 844 (96.2%) had informal training outside regulatory oversight during or after induction into the profession. The most common diseases encountered by them were common cold, pneumonia, and diarrhoea. 583 (66.5%) RMPs did not dispense any antibiotic for common cold symptoms. 59 (6.7%) and 64 (7.3%) of them could identify all main symptoms of pneumonia and diarrhoea, respectively. In pneumonia, 28 (3.2%) RMPs dispensed amoxicillin as first-line treatment, 819 (93.4%) dispensed different antibiotics including ceftriaxone, 721 (82.2%) dispensed salbutamol, and 278 (31.7%) dispensed steroid. In diarrhoea, 824 (94.0%) RMPs dispensed antibiotic, 937 (95.4%) dispensed ORS, 709 (80.8%) dispensed antiprotozoal, and 15 (1.7%) refrained from dispensing antibiotic and antiprotozoal together. Conclusions: Inappropriate diagnoses, irrational use of antibiotics and other drugs, and polypharmacy were