It has been previously reported that the improper prescribing of antibiotic eye drops is common among orthokeratology (ortho-k) practitioners. Guidelines have since been developed and disseminated to improve their understanding and implementation of antibiotic prescriptions. This study aimed to investigate the influence of these guidelines on the knowledge, attitude, and prescribing habits of ortho-k practitioners by means of a questionnaire, which was administered nationwide via an official online account to eye care practitioners (ECPs) involved in ortho-k lens fitting, 548 of whom completed the survey. Differences in characteristics before and after the dissemination of the guidelines and between the groups were explored using χ2 tests. The relationship between prescribing habits and demographics was analyzed using stepwise logistic regression models. The implementation of the guidelines significantly improved the overall prescribing habits of ECPs (p < 0.001), especially for prophylactic antibiotic use before and after ortho-k lens wear (p < 0.001). Most ECPs who prescribed antibiotics properly displayed significantly better knowledge of correct antibiotic use, which in turn affected the compliance in their ortho-k patients (p < 0.001). The ECPs’ occupations (professionals other than ophthalmologists and optometrists, including nurses and opticians), clinical setting (distributor fitting centers), and age (younger than 25 years) were risk factors for the misuse of antibiotics. Although the implementation of the antibiotic guidelines significantly improved overall prescribing habits, some practitioners’ prescribing behavior still needs improvement. A limitation of this study was that all questions were mandatory, requiring ECPs to recall information, and therefore was subjected to selection and recall bias.