The council refused the plans - which included 350 holiday lodges and a hotel - because of an "unacceptable" impact on the environment.
Knaresborough Town Council also complained there was "no proven business case" for the abandoned golf course site where proposals for 2,750 homes were previously rejected in a decision which wound up in a legal battle over the district's Local Plan.
A 300-bedroom hotel was also previously planned for the site, but these separate proposals never materialised.
Flaxby Park Ltd now has an option to appeal the latest decision against the eco-resort. It said it "remains confident that the proposal is appropriate for the site" and that it is now "considering the appropriate course of action".
A company spokesperson said: "Flaxby Park Ltd is extremely disappointed by the council’s decision to refuse the outline planning permission, on a site which was previously an established permitted leisure destination.
"The proposal is to provide a development which sits comfortably within the natural environment, providing a ‘getaway destination’ that is rural in feel but with easy access from main urban areas.
"The driving principle of the resort is to allow visitors to reconnect with nature within an ecologically rich environment involving the creation of new water bodies and woodlands."
The eco-resort proposals also included an outdoor swimming pool, spa and sports area, pub/restaurant and shops for the former golf course which closed in 2014.
The site sits alongside the A1(M) and another concern raised by the council was what it described as "constant traffic noise" from the motorway.
In response, Flaxby Park Ltd said while traffic noise may be audible in some areas of the site, the presence of a "significant tree belt would minimise the impact."
It added: "This was accepted by Harrogate Borough Council environmental protection who raised no objection subject to the imposition of suitably worded planning conditions.
"The proposed development would minimise the resort’s carbon footprint and provide a unique lodge development within the Yorkshire area.
"The proposal would also generate significant public benefit locally in the form of economic benefits with respect to increased visitor expenditure and the creation of employment opportunities."
In a decision notice, the council said the scale and layout of the eco-resort were "considered to have an unacceptable adverse impact upon the district's natural and historic environment".
It also said the proposals would cause harm to the Grade II listed Temple of Victory momentum which sits in the grounds of the nearby Allerton Park.
These are the reasons for refusal which could be examined by a government planning inspector if Flaxby Park Ltd decides to once again launch a legal challenge against the council.
By Jacob Webster, Local Democracy Reporter