Endoperoxide-containing molecules based on the antimalarial drug artemisinin have demonstrated various biological properties, including modulation of calcium homeostasis. This study evaluated the toxicity and osteogenic activity of five newly developed tetraoxanes (YB1, YB9, YB11, YB17 and T2), alongside three of their non-peroxidic analogues (IC22, IC26 and IC33), in zebrafish (Danio rerio) larvae. For each compound, LC₅₀ values were first determined. Behavioural responses and morphological alterations were studied as indicators of toxicological impact. The osteogenic activity was assessed through the operculum assay, followed by the analysis of gene expression markers related to calcium homeostasis (atp2a1), oxidative stress (sod1, cat), and osteogenesis (sp7, oc2). All the compounds evaluated induced an inhibition of osteogenic activity. T2, YB11, IC33 and IC26 affected the locomotor function by decreasing swimming activity. IC26 and IC33 induced morphological toxicity, characterized by a curved trunk and alterations in larval body curvature. From all the compounds studied, YB1, YB9, YB17 and IC22 showed selective anti-osteogenic activity, without displaying significant behavioural or morphological toxicity. In conclusion, the presence of a peroxide bond in the molecular structure of the compounds increases the anti-osteogenic activity at lower concentrations. All evaluated compounds exhibited anti-osteogenic activity and can be regarded as anti-osteogenic agents. However, YB17 did not induce transcription alterations in the genes analyzed and may thus represent the most promising compound in conditions where a controlled inhibition of bone formation is desirable.