Landraces are considered a crucial component of biodiversity conservation, serving as a reservoir of genetic diversity. Consequently, the collection, cultivation, and detailed characterization of such landraces constitute an inherent aspect of the world’s natural resource heritage. This effort holds promise for the development of elite varieties capable of thriving amidst continuous global climate fluctuations. In this context, we conducted a comprehensive assessment of the main agronomic attributes, physico-chemical properties, and functional quality traits of the major hot pepper landraces adapted to diverse climatic conditions in Tunisia. These landraces include ‘Dhirat’, ‘Semmane’, ‘Beldi’, ‘Nabeul’, ‘Jerid’, ‘Mahdia’, ‘Cayenne’, ‘Kairouan’, and ‘Baklouti’. Most of the pepper landraces exhibited satisfactory yields, ranging from 1163.25 to 1841.67 g plant−1 in ‘Jerid’ and ‘Kairouan’, respectively, indicating robust productivity, especially under prevailing climatic changes and high temperatures during both growing cycles. The levels of antioxidants comprising capsaicinoids, carotenoids, phenolics, and tocopherols, as well as radical scavenging activity, emerged as key discriminating factors among pungent pepper landraces. Irrespective of genotype, capsaicin and dihydrocapsaicin constituted the major capsaicinoids, accounting for 44–91% of the total capsaicinoids content. Total capsaicinoids ranged from 1.81 µg g−1 fw to 193.71 µg g−1 fw, with ‘Baklouti’ and ‘Jerid’ identified as the most pungent landraces. Total carotenoids ranged from 45.94 µg g−1 fw to 174.52 µg g−1 fw, with ‘Semmane’ and ‘Jerid’ exhibiting the highest levels. Considerable variation was observed in β-carotene content, spanning from 3% to 24% of the total carotenoids. α-Tocopherol content ranged from 19.03 µg g−1 fw in ‘Kairouan’ to 30.93 µg g−1 fw in ‘Beldi’, exerting a notable influence on the overall tocopherol content. Conversely, the β- and γ-tocopherol isomers were detected at very low concentrations. The total vitamin C content ranged from 132 mg 100g−1 fw in ‘Mahdia’ to 200 mg 100 g−1 fw in ‘Nabeul’, indicating relatively low genetic variability. However, large variability was detected in total phenolics content, ranging from 168.58 mg GAE kg−1 fw in ‘Beldi’ to 302.98 mg GAE kg−1 fw in ‘Cayenne’. Landraces such as ‘Dhirat’, ‘Nabeul’, ‘Semmane’, ‘Kairouan’, ‘Cayenne’, and ‘Mahdia’ appear suitable for both fresh consumption and processing, owing to their favorable average fruit weight, soluble solids content, and bioactive content. Among the pepper landraces tested, ‘Cayenne’ achieved the highest value of radical scavenging activity in both hydrophilic and lipophilic fractions (RSAHF and RSALF), with variations ranging from 59% to 120% for RSAHF and from 4% to 63% for RSALF. This study aims to preserve and enhance the value of local genetic resources and contribute to identify desirable traits for incorporation into breeding programs to develop high-quality, high-yielding landraces and elite lines.