Autism Spectrum Disorder (ASD) involves a complex of neurodevelopmental disorders with gastrointestinal co-morbidities appearing in approximately 70% of the population. This study seeks to analyze the gastrointestinal pathogenic and inflammatory status in pediatric subjects with ASD. To achieve this, biomarkers of nonspecific inflammatory activity such as calprotectin, lactoferrin, fecal pH and reducing sugars were analyzed in faces and fecal occult blood test (FOBT) was performed; anti-Saccharomyces cerevisiae antibodies (ASCAs) were also detected and measured in blood serum. A wide battery of bacterial, parasitic and fungal pathogens with clinical importance was researched in both study groups, the problem group (children with ASD) vs the related control group (Siblings of children with ASD without the disorder). Individuals with ASD had abdominal pain, loss of appetite, belching, flatulence, hard and lumpy stools, constipation and pushing in more than 50% of the population; no enteropathogenic bacteria was isolated as well as pathogenic fungi. However, opportunistic fungi were isolated in 41% of the problem group and all the species identified belonged to the Candida genus, which can damage the epithelial barrier of the intestine and induce epithelial inflammatory responses with cellular damage. Intestinal parasites were identified in 41% of ASD’s and 36% had positive fecal occult blood indicating damage to the intestinal mucosa, although its correlation with calprotectin was not statistically significant (p > 0.4909). Elevated levels of calprotectin were recorded in 75% of ASD individuals and 25% had elevated levels of ASCAs. These results show inflammatory alterations in the intestinal mucosa. Therefore, we suggest endoscopic and biopsy studies to determine the type of histological alteration and its etiology.