Colorectal cancer (CRC) is one of the most common cancers worldwide and a significant global health issue. The human gut microbiota, a complex ecosystem hosting numerous microorganisms such as bacteria, viruses, fungi, and protozoa, plays a crucial role. Increasing evidence indicates that gut microbiota is involved in CRC pathogenesis. In this study, the gut microbiota profiles, short-chain fatty acids, zonulin, and lipopolysaccharide-binding protein levels of newly diagnosed CRC patients were analyzed along with healthy controls to elucidate the relationship between CRC and the gut microbiota. The study included 16 newly diagnosed CRC patients and 16 healthy individuals. For microbiota analysis, DNA isolation from stool samples was performed using the Quick-DNA™ Fecal/Soil Microbe Miniprep Kit followed by sequencing using the MinION device. Data processing was conducted using Guppy software (version 6.5.7) and the Python (3.12) programming language. ELISA kits from Elabscience were utilized for analyzing LBP and zonulin serum levels. Fecal short-chain fatty acids were analyzed using GC-MS/MS equipped with a flame ionization detector and DB-FFAP column. Microbial alpha diversity, assessed using Shannon and Simpson indices, was found to be lower in CRC patients compared to healthy controls (p = 0.045, 0.017). Significant differences in microbial beta diversity were observed between the two groups (p = 0.004). At the phylum level, Bacteroidota was found to be decreased in CRC patients (p = 0.027). Potential biomarker candidates identified included Enterococcus faecium, Ruminococcus bicirculans, Enterococcus gilvus, Enterococcus casseliflavus, Segatella oris, and Akkermansia muciniphila. Serum zonulin levels were higher in CRC patients (CRC = 70.1 ± 26.14, Control = 53.93 ± 17.33, p = 0.048). There is a significant relationship between gut microbiota and CRC. A multifactorial evaluation of this relationship could shed light on potential biomarker identification and the development of new treatment options for CRC.