It is unknown whether diet quality is associated with microvascular structure. The present study aimed to investigate the relationship between diet quality, reflecting adherence to dietary guidelines, with retinal microvascular calibre in older adults. The dietary data of 2720 Blue Mountains Eye Study participants, aged 50+ years, were collected using a semi-quantitative FFQ. A modified version of the Healthy Eating Index for Australians was developed to determine total diet scores (TDS). Fundus photographs were taken and retinal vascular calibre measured using computer-assisted techniques and summarised. After adjusting for age, sex, BMI, mean arterial blood pressure, smoking, serum glucose, leucocyte count and history of diagnosed stroke or CHD, persons with higher TDS had healthier retinal vessels cross-sectionally, with wider retinal arteriolar calibre (by approximately 3 μm, comparing the highest with the lowest quartile of TDS, Ptrend= 0·0001) and narrower retinal venular calibre (by approximately 2·5 μm; Ptrend= 0·02). In younger subjects aged ≤ 65 years, increasing TDS (lowest to the highest quartile) was associated with healthier retinal vessels: approximately 4·4 μm wider retinal arteriolar (Ptrend< 0·0001) and approximately 2·3 μm narrower venular calibre (Ptrend= 0·03). After multivariable adjustment, however, baseline TDS were not associated with retinal arteriolar (Ptrend= 0·89) or venular calibre (Ptrend= 0·25), 5 years later. Also, baseline TDS were not associated with the 5-year change in retinal arteriolar (β = 0·14; P= 0·29) or venular calibre (β = − 0·26; P= 0·07). Greater compliance with published dietary guidelines (higher diet quality) was cross-sectionally associated with wider retinal arterioles and narrower venules, indicating better retinal microvascular health.