Dental plaque is a major etiological factor in the development of dental caries and periodontal diseases, making effective plaque control essential in children. With increasing environmental concerns, biodegradable bamboo toothbrushes have emerged as a sustainable alternative to conventional plastic toothbrushes; however, evidence comparing their clinical efficacy in pediatric populations remains limited. This crossover in vivo study evaluated and compared the plaque removal efficacy of plastic and bamboo manual toothbrushes in 44 healthy school-going children aged 8–12 years. Participants used a plastic toothbrush for two weeks, followed by a one-week washout period and subsequent use of a bamboo toothbrush for another two weeks. Plaque scores were assessed before and after each intervention using the Turesky-Gilmore-Glickman modification of the Quigley-Hein Plaque Index, and user preference was evaluated through a subjective questionnaire. Both toothbrushes demonstrated significant reductions in plaque scores, with plastic toothbrush scores decreasing from 1.76 ± 0.37 to 0.77 ± 0.28 and bamboo toothbrush scores from 1.93 ± 0.35 to 0.73 ± 0.28 (p < 0.001), with no significant difference between the two groups after intervention (p = 0.680). Subjective evaluation revealed that 85% of participants reported a better brushing experience with bamboo toothbrushes, while 65% preferred them over plastic toothbrushes. Within the limitations of the study, bamboo toothbrushes were found to be as effective as conventional plastic toothbrushes in plaque removal while demonstrating high user acceptability, supporting their use as an environmentally sustainable alternative for maintaining oral hygiene in children.