Fabrics serve as fomites in spreading nosocomial infections. As a patient is in close contact with bedsheets, it is important to assess the seasonal variation in bacterial diversity on these in healthcare units. The study was conducted to characterise the bacterial diversity on patients’ bedsheets across 7 months in a primary healthcare unit. Polyester-cotton blend fabric was stitched on bedsheets, and temporal dynamics of bacterial communities was assessed from May to November 2019. qPCR and amplicon sequencing of 16S rRNA gene was performed for profiling of bacterial community. Results revealed the dominance of Bacillota followed by Pseudomonadota, and Actinomycetota. A seasonal variation was observed in the bacterial load, with maximum values in June. This indicates the impact of environmental conditions on bacterial abundance and composition on fabrics in healthcare unit. The presence of priority pathogens on the patient bedsheets is a human health concern reiterating the need for season-specific laundering protocol.