Sepsis is one of the leading causes of morbidity and mortality worldwide, and culture-negative sepsis, despite its prevalence, is largely understudied. The current study intends to examine clinical characteristics and biomarkers in culture-positive and culture-negative sepsis, focusingon 30-day mortality and duration of hospital stay in both groups. Materials and methods: A prospective observational comparative cohort study was done on 150 patients admitted to theintensive care unit (ICU) and wards of Jaipur Golden Hospital. Patients with documented fungal, viral, or parasitic infections, as well as those who had undergone surgery or experienced trauma, were excluded. Results: The mean age of the patients was 51.31±18.94 years. Of150 patients, 95 (63.3%) were culture-negative, whereas 55 (36.7%) were culture-positive, with more men in the former and more women in the latter. Patients with negative cultures had fewer comorbidities. The levels of procalcitonin (PCT), C-reactive protein (CRP), and serum lactate were within the prescribed limit for both culture-negative and positive patients. A higher proportion (87.3%) of the organisms isolatedfrom culture-positive individuals were gram-negative, with Escherichia coli (E. coli) having the highest prevalence (27.3%), followed by Klebsiella (20%). There were 12.7% gram-positive isolates. The culture-negative patients had significantly better outcomes (P=0.003) as well as the duration of hospital stay (P<0.001) than the culture-positive patients. Culture-positive patients had a more severe illness, a higher incidence of septic shock, and a higher fatality rate than culture-negative patients. It can be concludedthat CRP and PCT can be used as clinically reliable sepsis biomarkers in both culture-positive and culture-negative patients. The study found that culture-negative sepsis is more prevalent and that there are substantial differences between culture-negative and culture-positive sepsis, with the former group having fewer comorbidities, less severe illness, a shorter duration of hospital stays, lower death rates, and better outcomes.