Muscarinic-cholinergic antagonists, such as atropine, are widely used to treat myopia, yet their mechanism of action remains unclear. Recent chick studies suggest atropine may act via a non-muscarinic pathway, specifically antagonism of serotonergic signalling. This study investigates the conservation of this mechanism across different models of myopia (form-deprivation myopia (FDM) and lens-induced myopia (LIM)), and across different muscarinic agents (i.e., pirenzepine). We also examined the role (and ocular distribution) of serotonergic signalling in myopia onset. Chicks undergoing FDM or LIM received daily injections of atropine (0.15 nmoles) or pirenzepine (1 µmole), alone or with serotonin (0.5 or 5 nmoles). To investigate serotonergic signalling in experimental myopia, chicks undergoing FDM and LIM were treated with serotonergic antagonists (mianserin (2 nmoles) or methiothepin (1.5 nmoles)). Ocular growth was measured via axial length and refraction. Serotonin levels, receptor binding, and synthesis activity were assessed using mass spectrometry, and transcriptomic profiling was used to map serotonergic components throughout the eye. Serotonin co-administration reduced the anti-myopic effects of atropine and pirenzepine by ∼70 %. Both FDM and LIM were associated with increased retinal serotonin receptor binding, which was blocked by muscarinic antagonists. Direct antagonism of serotonin receptors inhibited myopia development. Transcriptomic and biochemical analyses revealed widespread serotonergic signalling throughout the eye, limiting identification of a specific site of action. These findings suggest elevated serotonergic activity contribute to myopia progression and that muscarinic antagonists may exert their effects, at least in part, via modulation of serotonergic pathways.