ASD is one of the most heterogeneous and diagnostically difficult neurodevelopmental disorders seen in paediatric practice, with the main features being significant impairments in reciprocal social interaction, verbal and nonverbal communication, and stereotyped, inflexible patterns of behaviour. A variety of genetic, environmental and neurobiological factors have been investigated with regard to its etiology, but no single mechanistic explanation of ASD has been found. Objectives: This study aimed to extensively characterize a focused panel of biochemical, neuroendocrine and immune parameters in a paediatric ASD population to better understand their possible contributions to disease initiation and progression. The study was conducted with a case-control design, and included 80 children aged 6 to 12 years, of which 40 had a validated diagnosis of ASD and 40 were neurotypical children matched for age. Serum was obtained from peripheral venous blood samples from each subject, and quantitative assays conducted for the following analytes: GABA, dopamine, serotonin, BDNF, NGF, IL-6, TNF-α, TSH, FT3, and FT4. Results: The children with ASD had significantly lower levels of both GABA and dopamine in their serum compared to healthy controls, while the levels of serotonin were surprisingly higher to a statistically significant extent. Significant upregulation of both neurotrophins, BDNF and NGF, was observed in the ASD group. Immunological profiling showed a significant and large increase in the level of TNF-α, while there was no significant difference in the level of IL-6 between the two groups. The thyroid hormone evaluation did not show any significant difference in TSH or FT3 levels, although the FT4 levels were significantly reduced in the autistic group compared to the healthy group. Conclusions: All these concurrent dysregulations suggest that ASD pathophysiology is driven by the interplay of several biological axes, and not a single molecular defect. The biochemical changes described herein could not only provide a better understanding of the mechanism of ASD but could also be used to develop reliable peripheral biochemical markers that can be used for early diagnosis and future pharmacological targets.