Green nanotechnology plays a significant role in many domains, from medicine to agriculture. Antibiotic resistance has emerged in organisms because of the continuous and indiscriminate application of various antibiotics. MDR (Multi drug resistant) bacteria, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus were found to be resistant to beta- lactam antibiotics. In this study, the aqueous extract of Colletotrichum siamense was used to engineer nanoparticles (CsNps) and tea tree oil-based nanoemulsion (CsNe). Engineered CsNps and CsNe were subjected to biophysical characterization by using UV visible spectroscopy, FESEM, EDAX, FT-IR, HRTEM, SAED, and zeta potential to determine the charge, size, stability, elemental composition, topography, etc., The CsNps and CsNe were screened to detect antimicrobial activity by the zone of inhibition, MIC, MBC, and biofilm inhibition to its test potential against pathogenic E. coli, K. pnuemoniae, A. baumannii, and S. aureus. In silico studies were performed to study the binding affinity of the virulence proteins of MDR pathogens with secondary metabolites of Colletotrichum siamense. The results of both in vitro and in-silico studies of nanoparticles and nano-emulsion were observed to be more promising than beta-lactam antibiotics. Hence, in near future, these endophytic fungi, C. siamense mediated nanoparticles (CsNps) and nanoemulsion (CsNe) can be employed to develop an alternative to beta lactam antibiotics against the MDR pathogens.