Background Mucormycosis, attributed to a group of molds known as mucormycosis, is a rare yet life-threatening fungal infection often colloquially referred to as black fungus. While its incidence notably surged during the second wave of COVID-19 infections in India, it's essential to recognize that mucormycosis was a significant concern even before the advent of the pandemic. Understanding the prevalence and characteristics of this infection in the pre-COVID era provides a crucial context for evaluating its impact and dynamics during the pandemic. Multiple diagnostic methods, such as potassium hydroxide (KOH) mount, culture, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and histopathological examination (HPE), are available for identifying this lethal infection. The primary objective of this study is to ascertain the sensitivity of various diagnostic methods for mucormycosis and to analyze the comparative effectiveness of microbiological versus histopathological diagnoses. Methods We conducted a retrospective observational study spanning six months, from May 2021 to October 2021, encompassing all mucormycosis cases meeting the inclusion criteria and diagnosed via histopathological examination (HPE) in the departments of pathology and microbiology. Microbiological tests were performed prior to the histopathological examinations. Sensitivity was assessed through statistical analysis, and the relationship between microbiological and histopathological diagnoses was evaluated using the chi-square test. Results Biopsy samples of 77 patients were collected, comprising 56 male and 21 female patients. Regarding age distribution, most patients fell within the 41-60 age bracket, while the smallest proportion was over 60 years old. The sensitivity and specificity of histopathological diagnosis, confirmed with periodic acid-Schiff (PAS) and Grocott-Gomori's methenamine silver (GMS) staining, both recorded a flawless 100%. KOH mount sensitivity stood at 88.3%, while fungal culture and MALDI-TOF exhibited sensitivities of 75.3%. Histopathological analysis revealed that 17% of cases displayed minimal fungal hyphae alongside necrotic tissue, whereas 58% exhibited abundant fungal hyphae accompanied by inflammatory cells. Additionally, absolute neutrophilia was observed in 55% of cases. Conclusions In our study, histopathology and KOH mount emerged as not only compassionate but also cost-effective diagnostic tools for identifying mucormycosis. The economic aspect of these diagnostic methods is highlighted in the results section to provide a comprehensive understanding of their cost-effectiveness. Additionally, we utilized MALDI-TOF MS as a straightforward, economically viable, and expeditious method specifically for confirming the fungal subtype in mucormycosis cases. The rationale behind choosing either MALDI-TOF MS or KOH for the diagnosis is elucidated, contributing to a clearer interpretation of our diagnostic approach. Furthermore, our findings indicate that absolute neutrophilia consistently manifests in 55% of mucormycosis cases.