Sunamganj is a prospective area for layer farming where many smallholders are involved in layer production. We hypothesized that these layer smallholders’ adoption levels of biosecurity varied significantly. Therefore, we designed a cross-sectional survey to know the adoption level of biosecurity practices by small-scale layer farmers in the Sunamganj district. We collected data through face-to-face interviews of randomly selected 45 layer farmers from the study area using a questionnaire containing 48 biosecurity-related questions. From the data, we calculated the biosecurity adoption index (AI), a simple scoring system to determine the adoption level of biosecurity by the smallholders. We categorized smallholders as low, partial, and high adopters based on the AI value. A two-sample t-test and one-way ANOVA measured variations in the mean AI value between levels of demographic factors to identify key predictors. The study revealed that only 17.78% (8/45) of the smallholders were high adopters, while 57.76% (26/45) were partial adopters, and the rest, 24.44% (11/45) were low adopters. The overall mean AI value was 58.75±21.26 in the survey. Farming experience, family members in the farming, presence of labour, income, and holding type were significant (p<0.05) key predictors that influenced the AI. This study underlines the scenario of adopting biosecurity practices and their plausible drivers. Results of the present study could assist respective authorities in taking evidence-based necessary steps to improve biosecurity practices in the future.