Natural decomposition of sugarcane trash is slow, taking more than three months, and hence, it becomes a problem with inter-cultivation and restricts the growth of the ratoon of the sugarcane crop. Trash blanketing is a common management practice carried out but does not solve the above problems. This study investigates the potential of using selected microbial combinations, including fungal-bacterial biofilms (FBB), for rapid decomposition of sugarcane trash. Two studies were carried out at laboratory level, namely, to identify the sugarcane trash decomposition process and then to evaluate selected microbial combinations in enhancing sugarcane trash decomposition. In the first study, sugarcane trash samples were placed in 24 wells of a tissue culture plate separately, and evaluated the surface functional groups of organic compounds by Fourier transform infrared (FTIR) spectroscopy. In the second study, 24 treatments in a completely randomised design were used to identify selected microbial combinations that could be effective in sugarcane trash decomposition. When considering the results of the first study, there was a positive correlation between weight loss and FTIR peak degradation of organic molecular functional groups, particularly O-H of the carboxylic group, C-H of the aromatic methyl group, and Si-O of the cuticle wax layer. The results of the second study showed that urea being a chemical treatment, was significantly effective in reducing the C/N ratio of decomposed trash. In the microbial treatments, bacterium B2 (yet to be identified) was effective on trash fragmentation, and the FBB coded as F1F2B1B2 was effective on both trash fragmentation and reducing its C/N ratio. The combination should therefore be tested for trash decomposition in the long run of the sugarcane crop cycle under field conditions.